The objective of this proposal is to apply novel affinity mass spectrometry (AMS) technologies to identify protein biomarker fingerprints indicative of alcohol consumption. The methodology used combines micro-affinity protein capture from complex biological solutions with mass spectrometric detection. In this first phase (R43), we will screen a small number of plasma samples (n=48) to identify candidate protein profiles, that can appropriately differentiate sample origin from healthy abstinent controls from individuals differentially exposed to alcohol. These samples will be screened with affinity pipettes employing numerous wide specificity affinity surfaces, which include hydrophilic, hydrophobic and metal chelating ligands to name a few. Each of these ligand surfaces possesses specific affinity characteristics that will result in the capture and purification of various protein groups. Observed molecular profiles (fingerprints) that have statistically significant features that correctly group samples will be viewed as potential biomarkers. In the second (validation) phase (R44), we will construct immuno-targeted assays for identified significant features determined within each biomarker fingerprint and individually validate them against a larger number of samples (n=400). In the event that multiple protein biomarkers are validated, a multiplexed immuno-based AMS panel for alcohol exposure will be developed. This will be achieved through the construction of [unreadable] multi-analyte affinity pipettes that will selectively retrieve the targeted biomarkers that differentiate healthy from diseased states. Such panels will also be subject to the same validation process that each of its components individually endured. The ultimate result of this research will be a validated protein biomarker assay(s) that can be used to screen for alcohol exposure. [unreadable] [unreadable] [unreadable]